Good. Are you going to do FPLC again? -UM
Week 11 and 12:
FPLC result for Elution 1 in a buffer containing 50mM Tris, 150mM NaCl, and 1mM DTT
FPLC separates the proteins in elution 1 based on size, however no distinct peak was recorded at 35kDa that would indicate the presence of the STPP protein. It may have dimerized and resulted in the peak at about 80 kDa before the first standard.
Elution 1 concentrated to 1.46mg/ml measured at 280nm
In order to get a higher concentration of the protein present in elution 1, the sample was concentrated to 1.46mg/ml using a sartorius stedim concentrator with a 10000 MWCO PES membrane.
Elution 1 before concentrating containing .47mg/ml measured at 280nm
SDS-Page gel stained with Imperial Stain. Well 1 ColorPlus protein ladder, Sample 0 is cell lysate after induction, sample 2 is the soluble fraction, sample 3 is flow through, sample 4 is wash, sample 5 is elution 1, and sample 6 is elution 2.
The SDS-Page gel shows that elution 1 does have a band of protein around 30kDa, but it also contains contamination bands. STPP has a weight of about 35kDa, so this may not be the target protein. FPLC will hopefully further purify the protein.
500ml culture of Bl21(DE3) cells with the STPP insert was grown to log phase then induced with 260ml of IPTG.
Try uploading your images again, they aren't showing on the page. Include your cloning results too. Good analysis. -UM
Week 9 and 10
(A second round of cloning was done at the same time as sequencing the first round. Round 1 yielded a successful clone while round 2 was unsuccessful at producing transformed colonies. Will upload pictures of master plate and unsuccessful round 2 plates once I get into lab.)
BLAST alignment comparing codon optimized STPP CDS as query, and synthesized forward and reverse read combination from sample colony 4 as subject. 100% homology no indel.
After comparing the results of the 8 samples sent for sequencing against the CDS for the target protein using BLAST, it was determined that colony 4 contained positive clones. Below are the sequencing results from the core for the forward and reverse reads on sample 4. N's stand for base pairs that were not able to be identified by the colorimetric sequencing system. To determine these base pairs, the reverse read was reverse complimented and combined with the forward read. When combined, there was a base determined for every single position. This sequence was then compared against the optimized CDS for the target protein STPP with 100% homology and 100% query coverage.
Reverse DNA Sequencing result for colony 4 positive clone:
NNNNNNNNNNNNNNNGNNCTNNGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAA
TTCGGATCCGTATCCACCTTTACTGTTACAGAAAGTAGTCCGGAGTCTTTTTAGAAACTTGTGCCTCGCCACGACGA
GGTGCAGGGTCGAACTGAAAGAAGCACTTATTCATGTGTTCGTCCAGCTCCAGCAGACCCGCGAGATTGCCACAACG
GTAGCAGTAGTTAGGCGCAGAGAAGATCGTAACCAGCTGATCCTGGTGGGTCCAAGAATAACCGTCCATAACGAGCT
GATGAGCACGCGCGATGGTTTTGATTTTGTTGTTGTGACAGAAACCCTCGGTAACACCCTGGCCGAAGGTAAAGCCC
GCACCACGCGGGGAGATACCCCAACCGTCACGGTCGTCCGGGTCAGACCAGAGCAGGTCACACATCGGACCCTCGTG
CGGAACTTCCTGAACACGGTCCAGATTACGGATGTGAGAGAAGGTGTCAACGGTCGGAGAGAGACCACCGTGCAGGC
AGAAAATGTCGTTTTCAACCAGCGCGGTCAGCGGCAGGTAGTCGAACAGGTCGGTGAAGATGGTCCAAACGTTCGCA
GAGCCGTATTTACGAATACATTCGTCGTAGAAGCCGTAAACCTGGGTGATCTGACGAGACTCATGGTTACCACGCAG
CAGATGCAGACGTTGCGGGTAGCGCAGTTTGTACAGGAGCANGAGAGTAACGGTTTCAACAGAGTAGTAACCACGGT
CAACGTAGTCACCCATGAACAGATAGTTGGTGTCCGGTGGGAGGCCACCGATTTTGAAGAGTTCCAGGAGGTCGTGG
AACTGGNCGTGAACGTCACCGCAAACGGTCACCGGCGCACGAACGGCGTGGACATTGTTTTCTTTTTCCAGTACTTC
TTTAACTTTTTCGCACAGGCGCGCNACCTGNTGTTCAGACAGCNGGTTTGCACTGGNNANATAGTNGATCATTTCGT
CNANGTCCAGCGCANANNNNNNAACNNNNANNNNNNCCCNNANNGGNCATGGNATNGAGNACAGGTNNNCGGNNCCN
NATCTANNNNNANANGATGATGANGANGNNGCANANGGNNNATCNCNNNNNNNNNNNNNAANNNTNNTNNNNGGNNN
NNTNTCNNNNNANNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNANNNNTNNCNNNNATNNGNNNNNNNNN
NNNNNNNNCCNN
Forward DNA Sequencing result for colony 4 positive clone:
NNNNNNNNNNNNNNNTTTAGNNGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTAC
CGAGAACCTGTACTTCCAATCCATGACCACTGCGGGTGGTGGTTCTGCGGTTGGTTCTTCTTCTGCGCTGGACCTCG
ACGAAATGATCAACTATGTTATCCAGTGCAAACCGCTGTCTGAACAACAGGTTGCGCGCCTGTGCGAAAAAGTTAAA
GAAGTACTGGAAAAAGAAAACAATGTCCACGCCGTTCGTGCGCCGGTGACCGTTTGCGGTGACGTTCACGGCCAGTT
CCACGACCTCCTGGAACTCTTCAAAATCGGTGGCCTCCCACCGGACACCAACTATCTGTTCATGGGTGACTACGTTG
ACCGTGGTTACTACTCTGTTGAAACCGTTACTCTCCTGCTCCTGTACAAACTGCGCTACCCGCAACGTCTGCATCTG
CTGCGTGGTAACCATGAGTCTCGTCAGATCACCCAGGTTTACGGCTTCTACGACGAATGTATTCGTAAATACGGCTC
TGCGAACGTTTGGACCATCTTCACCGACCTGTTCGACTACCTGCCGCTGACCGCGCTGGTTGAAAACGACATTTTCT
GCCTGCACGGTGGTCTCTCTCCGACCGTTGACACCTTCTCTCACATCCGTAATCTGGACCGTGTTCAGGAAGTTCCG
CACGAGGGTCCGATGTGTGACCTGCTCTGGTCTGACCCGGACGACCGTGACGGTTGGGGTATCTCCCCGCGTGGTGC
GGGCTTTACCTTCGGCCAGGGTGTTACCGAGGGTTTCTGTCACAACAACAAAATCAAAACCATCGCGCGTGCTCATC
AGCTCGTTATGGACGGTTATTCTTGGACCCACCAGNATCAGCTGGTTACNATCTTCTCTGCGCCTAACTACTGCTAC
CGTTGTGGCAATCTCGCGGGTCTGCTGGANCTGGACGAACACATGAATAAGTGCTTCTTTCAGTTCGACCNTGCNNN
NANCTCNNCGANAGCTNNGNNNNCTCNNNNNCCNCCNCNACCNCCNCTNNNATCNNCTGCNANNNNCNNAANNNNCT
NNNNNNNNCNNCNNCNNNTNNNNANACTANCNNANNCNNNNNNNNNNNNNNNNNNNNGNNNTTNNNNNNANNNNNNN
NNNNNNNNNNNNNNNTTNN
DNA sequencing results successfully available for all 8 samples with pLIC primers
The table above shows that results are available for each of 8 samples using pLIC forward and reverse primers.
Nanodrop results from 8 cultures made from 4 master colonies mini-prepped
Mini-prepping purifies the pNIC with gene insert after it has been cloned in DH5-Alpha cells. The first round of cloning yielded only four colonies, so 2 cultures were grown from each colony to be sent to DNA sequencing (one with a forward and one with a reverse primer. The above image is the nanodrop results after mini-prepping for these 8 cultures. They have excellent concentrations and no protein contamination because every sample has a 260/280nm ratio close to the optimum 1.9. There may be slight other contaminants resulting from the mini-prep steps as seen by the low 260/230nm ratios, namely in FOR2, FOR3, and REV4.
Nice captions and analysis. Could use more data, where is your gel pic? -UM
Week 7 and 8
Nanodrop image of cut pNIC-Bsa4 with absorbance of .403 at 230nm and a calculated concentration of 33.6ng/ul. 260/230nm ratio 1.67, and 260/280nm ration 1.86
After the pNIC-Bsa4 plasmids were midi-prepped, the SacB gene was cut out using the restriction enzyme BsaI-HF. A PCR clean up procedure was then carried out on these cut plasmids. The image shows a 160/180nm ratio of 1.86, which is near the optimum 1.9, so this suggests no protein contamination. However, the other value of 1.67 is not close to the optimum 2.1, which suggests other contaminants. These could be leftover restriction enzyme or a solution from the PCR clean up procedure.
Nanodrop image of pNIC-bsa4 midi-prepped. Calculated concentration of 48.1 ng/ul. Absorbance of 0.354 at 230 nm. 260/280nm ration of 1.99, 260/230nm ratio of 2.72
Midi-prepping the pNIC-Bsa4 was done to purify the plasmid and extract it from the DH5 alpha cells they were in. The above image shows the 260/280nm ratio to be 1.99. This number is close to the optimum value of 1.9, which suggests no protein contamination. However, the 260/230nm ratio of 2.72 suggests that there are other contaminants at the 230nm absorbance. This could be from parts of the DH5 Alpha cells not being fully removed during the midi-prepping process. The small yield could have been due to error in the midi-prep.
DH5 Alpha cells with pNIC-Bsa4 pellets from 160ml LB culture.
This image shows the resultant pellets from DH5 Alpha cells transformed with pNIC-Bsa4. The next step is to midi-prep these pellets to extract and purify the pNIC plasmid for use in Cloning.
Nice work, good captions and analysis. Keep it up! Week 5 & 6
PCR Clean Up
Nanodrop image of PCR clean up done with Sigma clean up kit on PCR squared product. Absorbance of 1.202 at 230 nm. 260nm/280nm ratio 1.86, 260nm/230nm ratio 2.23. Calculated concentration of 133.9 ng/ul
The Nanodrop image shows a gene concentration yield of 133.9 ng/ul. The 260nm/280nm ratio is 1.86 which is .04 away from the optimum value of 1.9, displaying almost no protein contamination. The 260/230 ratio is 2.23 is higher than the 2.1 desired value which may reflect some other contaminants at the 230nm absorbance.
PCR Squared
PCR squared gel check ran on 1% agarose TAE gel. Lane one shows a 1kb NEB nucleic acid ladder. Lanes 2, 3, 4, and 5 show PCR squared products with bands at about 1000 bp and 100bp. lane 5 shows distortion and assimilation into well 4
PCR squared is just a larger scale amplification of the secondary PCR product which is the desired gene sequence. The main band appears at the 1000kb gene length, however there are also light bands at about 100bp. These bands may be contamination, or more likely they are just a result of unbound primers in the solution. The same thermocycler settings were used as secondary PCR.
Due to human error, different voltages were applied to the gel. As a result of the gel running at too high of a voltage, the gel began to melt which led to wells 4 and 5 merging together. The voltage changes also are responsible for the warped bands.
Secondary PCR
Secondary PCR Gel check ran in 1% agarose TAE gel. Lane one shows a 100bp NEB nucleic acid ladder. Lane two shows secondary PCR product with band at 1000bp
Secondary PCR takes the primary PCR product and replicates only the full gene sequence, which is why only one band appears at 1000bp, the gene size. The slight streak up until the band may be a product of excess Etbr being pulled through the gel. The two designed tail primers had high melting temperatures of 70°C due to a high GC content. Normally the annealing temperature would be set at 5°C under this, but it is rather high so to ensure proper binding of primers a 58°C temperature was used.
Week 3 & 4
Daniel, - good work. For your gels that you share - don't show your partners lanes. (can edit the .png in PAINT). - Dr. B 092513 Primary PCR:
Primary PCR gel visualization in a 1% agarose TAE gel. Lane 1 is the primary PCR product and Lane 2 is an NEB 100bp DNA ladder
Results:
The first lane contains the PCR product of the optimized oligo-primer mix for the gene STPP. A smear appeared because there was nonspecific amplification of the primer segments, some are smaller and some may even be larger than the actual gene. The main portion of the smear spans 500-1000 base pairs.
Practice Restriction Enzyme Digest:
1% Agarose TAE gel visualized by EtBr .digest with 1kb NEB DNA ladder in lane 1, uncut plasmid in well 2, digestion by EcoRI in lane 3, digestion by PvuII in lane 4, and digestion by EcoRI and PvuII in lane 5. Lanes 6-10 used for another study
Practice RE digest with 1kb ladder on left, digestion with EcoRI in second lane, digestion with PvuII in third well, and digestion with EcoRI and PvuII in fourth lane
Results:
Lane 2 resulted in a smear because the plasmids coiled up and therefore ran at different speeds. Lane three showed successful digestion by EcoRI. PvuII cleaves the enzyme in 2 different locations, which explains the 2 bands in lane 3. When looking at the virtual gel, it can be seen that digestion by the enzymes EcoRI and PvuII together yields 3 bands. In the actual gel, only 2 bands are present. This occurred because the third band of DNA was too small so it ran off of the gel and could not be visualized.
Tail Primer Design: Forward Primer: 5’ TACTTCCAATCCATGACCACTGCGGGT 3’ 27 bp GC Content 51.9% 0 mM Mg2+ Tm 63.9 oC 1.5 mM Mg2+ Tm _70.5 oC 2 mM Mg2+ Tm 71.0 oC 4 mM Mg2+ Tm _72.1 oC 6 mM Mg2+ Tm _72.6 oC
Reverse Primer: 5’ _CTCCGGACTACTTTCTGTAACAGTAAAGGTGGATA_ 3’ Reverse complement it: 5’ TATCCACCTTTACTGTTACAGAAAGTAGTCCGGAG 3’ __35 bp GC Content 42.9% 0 mM Mg2+ Tm _61.5 oC 1.5 mM Mg2+ Tm _68.9 oC 2 mM Mg2+ Tm _69.4 oC 4 mM Mg2+ Tm _70.3 oC 6 mM Mg2+ Tm _70.7 oC
Practice PCR 1% TAE agarose gel with 100bp NEB ladder in lane 1. Lanes 2-5 contain .3ng, 3ng, 30ng, and 0 ng of pGBR22 DNA respectively. Lanes 6-9 were used for another study
Results:
Even though there were varying amounts of DNA in the four PCR samples, wells 2, 3, and 4 do not show an extreme difference in the brightness of the bands. This must mean that the PCR process does not replicate DNA in proportion to the initial DNA in the samples. Well 5 shows a light band of contamination right around the 1000bp range which is where the other bands are located. This is reasonable because pGBR22 has a length of around 1000bp.
Figure 1. pGBR22 DNA Nanodrop spectrophotometer result for trial one with concentration 153.3 ng/ul. 260/280 ratio value of 1.86, 260/230 ratio value of 2.05
The 260/280nm ratio is near 1.9, which suggests almost no protein contamination. The 260/230nm ratio is close to the desired 2.1, suggesting that there is little other contaminants at the 230nm absorbance.
Figure 2. pGBR22 DNA Nanodrop spectrophotometer result for trial one with concentration 151.8 ng/ul. 260/280 ratio value of 1.89, 260/230 ratio value of 2.09
The 260/280nm ratio is near 1.9, which suggests almost no protein contamination. The 2.09 value for the260/230nm ratio is close to the desired 2.1, suggesting that there is little other contaminants at the 230nm absorbance.
Daniel - results? - Dr. B 090913
Week 11 and 12:
FPLC separates the proteins in elution 1 based on size, however no distinct peak was recorded at 35kDa that would indicate the presence of the STPP protein. It may have dimerized and resulted in the peak at about 80 kDa before the first standard.
In order to get a higher concentration of the protein present in elution 1, the sample was concentrated to 1.46mg/ml using a sartorius stedim concentrator with a 10000 MWCO PES membrane.
The SDS-Page gel shows that elution 1 does have a band of protein around 30kDa, but it also contains contamination bands. STPP has a weight of about 35kDa, so this may not be the target protein. FPLC will hopefully further purify the protein.
500ml culture of Bl21(DE3) cells with the STPP insert was grown to log phase then induced with 260ml of IPTG.
Try uploading your images again, they aren't showing on the page. Include your cloning results too. Good analysis. -UM
Week 9 and 10
(A second round of cloning was done at the same time as sequencing the first round. Round 1 yielded a successful clone while round 2 was unsuccessful at producing transformed colonies. Will upload pictures of master plate and unsuccessful round 2 plates once I get into lab.)
After comparing the results of the 8 samples sent for sequencing against the CDS for the target protein using BLAST, it was determined that colony 4 contained positive clones. Below are the sequencing results from the core for the forward and reverse reads on sample 4. N's stand for base pairs that were not able to be identified by the colorimetric sequencing system. To determine these base pairs, the reverse read was reverse complimented and combined with the forward read. When combined, there was a base determined for every single position. This sequence was then compared against the optimized CDS for the target protein STPP with 100% homology and 100% query coverage.
Reverse DNA Sequencing result for colony 4 positive clone:
NNNNNNNNNNNNNNNGNNCTNNGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAA
TTCGGATCCGTATCCACCTTTACTGTTACAGAAAGTAGTCCGGAGTCTTTTTAGAAACTTGTGCCTCGCCACGACGA
GGTGCAGGGTCGAACTGAAAGAAGCACTTATTCATGTGTTCGTCCAGCTCCAGCAGACCCGCGAGATTGCCACAACG
GTAGCAGTAGTTAGGCGCAGAGAAGATCGTAACCAGCTGATCCTGGTGGGTCCAAGAATAACCGTCCATAACGAGCT
GATGAGCACGCGCGATGGTTTTGATTTTGTTGTTGTGACAGAAACCCTCGGTAACACCCTGGCCGAAGGTAAAGCCC
GCACCACGCGGGGAGATACCCCAACCGTCACGGTCGTCCGGGTCAGACCAGAGCAGGTCACACATCGGACCCTCGTG
CGGAACTTCCTGAACACGGTCCAGATTACGGATGTGAGAGAAGGTGTCAACGGTCGGAGAGAGACCACCGTGCAGGC
AGAAAATGTCGTTTTCAACCAGCGCGGTCAGCGGCAGGTAGTCGAACAGGTCGGTGAAGATGGTCCAAACGTTCGCA
GAGCCGTATTTACGAATACATTCGTCGTAGAAGCCGTAAACCTGGGTGATCTGACGAGACTCATGGTTACCACGCAG
CAGATGCAGACGTTGCGGGTAGCGCAGTTTGTACAGGAGCANGAGAGTAACGGTTTCAACAGAGTAGTAACCACGGT
CAACGTAGTCACCCATGAACAGATAGTTGGTGTCCGGTGGGAGGCCACCGATTTTGAAGAGTTCCAGGAGGTCGTGG
AACTGGNCGTGAACGTCACCGCAAACGGTCACCGGCGCACGAACGGCGTGGACATTGTTTTCTTTTTCCAGTACTTC
TTTAACTTTTTCGCACAGGCGCGCNACCTGNTGTTCAGACAGCNGGTTTGCACTGGNNANATAGTNGATCATTTCGT
CNANGTCCAGCGCANANNNNNNAACNNNNANNNNNNCCCNNANNGGNCATGGNATNGAGNACAGGTNNNCGGNNCCN
NATCTANNNNNANANGATGATGANGANGNNGCANANGGNNNATCNCNNNNNNNNNNNNNAANNNTNNTNNNNGGNNN
NNTNTCNNNNNANNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNANNNNTNNCNNNNATNNGNNNNNNNNN
NNNNNNNNCCNN
Forward DNA Sequencing result for colony 4 positive clone:
NNNNNNNNNNNNNNNTTTAGNNGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTAC
CGAGAACCTGTACTTCCAATCCATGACCACTGCGGGTGGTGGTTCTGCGGTTGGTTCTTCTTCTGCGCTGGACCTCG
ACGAAATGATCAACTATGTTATCCAGTGCAAACCGCTGTCTGAACAACAGGTTGCGCGCCTGTGCGAAAAAGTTAAA
GAAGTACTGGAAAAAGAAAACAATGTCCACGCCGTTCGTGCGCCGGTGACCGTTTGCGGTGACGTTCACGGCCAGTT
CCACGACCTCCTGGAACTCTTCAAAATCGGTGGCCTCCCACCGGACACCAACTATCTGTTCATGGGTGACTACGTTG
ACCGTGGTTACTACTCTGTTGAAACCGTTACTCTCCTGCTCCTGTACAAACTGCGCTACCCGCAACGTCTGCATCTG
CTGCGTGGTAACCATGAGTCTCGTCAGATCACCCAGGTTTACGGCTTCTACGACGAATGTATTCGTAAATACGGCTC
TGCGAACGTTTGGACCATCTTCACCGACCTGTTCGACTACCTGCCGCTGACCGCGCTGGTTGAAAACGACATTTTCT
GCCTGCACGGTGGTCTCTCTCCGACCGTTGACACCTTCTCTCACATCCGTAATCTGGACCGTGTTCAGGAAGTTCCG
CACGAGGGTCCGATGTGTGACCTGCTCTGGTCTGACCCGGACGACCGTGACGGTTGGGGTATCTCCCCGCGTGGTGC
GGGCTTTACCTTCGGCCAGGGTGTTACCGAGGGTTTCTGTCACAACAACAAAATCAAAACCATCGCGCGTGCTCATC
AGCTCGTTATGGACGGTTATTCTTGGACCCACCAGNATCAGCTGGTTACNATCTTCTCTGCGCCTAACTACTGCTAC
CGTTGTGGCAATCTCGCGGGTCTGCTGGANCTGGACGAACACATGAATAAGTGCTTCTTTCAGTTCGACCNTGCNNN
NANCTCNNCGANAGCTNNGNNNNCTCNNNNNCCNCCNCNACCNCCNCTNNNATCNNCTGCNANNNNCNNAANNNNCT
NNNNNNNNCNNCNNCNNNTNNNNANACTANCNNANNCNNNNNNNNNNNNNNNNNNNNGNNNTTNNNNNNANNNNNNN
NNNNNNNNNNNNNNNTTNN
Mini-prepping purifies the pNIC with gene insert after it has been cloned in DH5-Alpha cells. The first round of cloning yielded only four colonies, so 2 cultures were grown from each colony to be sent to DNA sequencing (one with a forward and one with a reverse primer. The above image is the nanodrop results after mini-prepping for these 8 cultures. They have excellent concentrations and no protein contamination because every sample has a 260/280nm ratio close to the optimum 1.9. There may be slight other contaminants resulting from the mini-prep steps as seen by the low 260/230nm ratios, namely in FOR2, FOR3, and REV4.
Nice captions and analysis. Could use more data, where is your gel pic? -UM
Week 7 and 8
Nice work, good captions and analysis. Keep it up!
Week 5 & 6
PCR Clean Up
PCR Squared
Due to human error, different voltages were applied to the gel. As a result of the gel running at too high of a voltage, the gel began to melt which led to wells 4 and 5 merging together. The voltage changes also are responsible for the warped bands.
Secondary PCR
Week 3 & 4
Daniel, - good work. For your gels that you share - don't show your partners lanes. (can edit the .png in PAINT). - Dr. B 092513
Primary PCR:
Results:
The first lane contains the PCR product of the optimized oligo-primer mix for the gene STPP. A smear appeared because there was nonspecific amplification of the primer segments, some are smaller and some may even be larger than the actual gene. The main portion of the smear spans 500-1000 base pairs.
Practice Restriction Enzyme Digest:
Results:
Lane 2 resulted in a smear because the plasmids coiled up and therefore ran at different speeds. Lane three showed successful digestion by EcoRI. PvuII cleaves the enzyme in 2 different locations, which explains the 2 bands in lane 3. When looking at the virtual gel, it can be seen that digestion by the enzymes EcoRI and PvuII together yields 3 bands. In the actual gel, only 2 bands are present. This occurred because the third band of DNA was too small so it ran off of the gel and could not be visualized.
Tail Primer Design:
Forward Primer:
5’ TACTTCCAATCCATGACCACTGCGGGT 3’ 27 bp
GC Content 51.9%
0 mM Mg2+ Tm 63.9 oC 1.5 mM Mg2+ Tm _70.5 oC 2 mM Mg2+ Tm 71.0 oC
4 mM Mg2+ Tm _72.1 oC 6 mM Mg2+ Tm _72.6 oC
Reverse Primer:
5’ _CTCCGGACTACTTTCTGTAACAGTAAAGGTGGATA_ 3’ Reverse complement it:
5’ TATCCACCTTTACTGTTACAGAAAGTAGTCCGGAG 3’ __35 bp
GC Content 42.9%
0 mM Mg2+ Tm _61.5 oC 1.5 mM Mg2+ Tm _68.9 oC 2 mM Mg2+ Tm _69.4 oC
4 mM Mg2+ Tm _70.3 oC 6 mM Mg2+ Tm _70.7 oC
>gi|124015065|gb|EF198106.1| Expression vector pNIC28-Bsa4, complete sequence
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGACCACTGCGGGTGGTGGTTCTGCGGTTGGTTCTTCTTCTGCGCTGGACCTCGACGAAATGATCAACTATGTTATCCAGTGCAAACCGCTGTCTGAACAACAGGTTGCGCGCCTGTGCGAAAAAGTTAAAGAAGTACTGGAAAAAGAAAACAATGTCCACGCCGTTCGTGCGCCGGTGACCGTTTGCGGTGACGTTCACGGCCAGTTCCACGACCTCCTGGAACTCTTCAAAATCGGTGGCCTCCCACCGGACACCAACTATCTGTTCATGGGTGACTACGTTGACCGTGGTTACTACTCTGTTGAAACCGTTACTCTCCTGCTCCTGTACAAACTGCGCTACCCGCAACGTCTGCATCTGCTGCGTGGTAACCATGAGTCTCGTCAGATCACCCAGGTTTACGGCTTCTACGACGAATGTATTCGTAAATACGGCTCTGCGAACGTTTGGACCATCTTCACCGACCTGTTCGACTACCTGCCGCTGACCGCGCTGGTTGAAAACGACATTTTCTGCCTGCACGGTGGTCTCTCTCCGACCGTTGACACCTTCTCTCACATCCGTAATCTGGACCGTGTTCAGGAAGTTCCGCACGAGGGTCCGATGTGTGACCTGCTCTGGTCTGACCCGGACGACCGTGACGGTTGGGGTATCTCCCCGCGTGGTGCGGGCTTTACCTTCGGCCAGGGTGTTACCGAGGGTTTCTGTCACAACAACAAAATCAAAACCATCGCGCGTGCTCATCAGCTCGTTATGGACGGTTATTCTTGGACCCACCAGGATCAGCTGGTTACGATCTTCTCTGCGCCTAACTACTGCTACCGTTGTGGCAATCTCGCGGGTCTGCTGGAGCTGGACGAACACATGAATAAGTGCTTCTTTCAGTTCGACCCTGCACCTCGTCGTGGCGAGGCACAAGTTTCTAAAAAGACTCCGGACTACTTTCTGTAACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAAT
Virtual Plasmids:
Week 1 & 2
Practice PCR:
Results:
Even though there were varying amounts of DNA in the four PCR samples, wells 2, 3, and 4 do not show an extreme difference in the brightness of the bands. This must mean that the PCR process does not replicate DNA in proportion to the initial DNA in the samples. Well 5 shows a light band of contamination right around the 1000bp range which is where the other bands are located. This is reasonable because pGBR22 has a length of around 1000bp.
Oligo-primer design:
1 ATGACCACTGCGGGTGGTGGTTCTGCGGTTG 31
2 CATAGTTGATCATTTCGTCGAGGTCCAGCGCAGAAGAAGAACCAACCGCAGAACCACCAC 60
3 CCTCGACGAAATGATCAACTATGTTATCCAGTGCAAACCGCTGTCTGAACAACAGGTTGC 60
4 TTCTTTTTCCAGTACTTCTTTAACTTTTTCGCACAGGCGCGCAACCTGTTGTTCAGACAG 60
5 AAAAAGTTAAAGAAGTACTGGAAAAAGAAAACAATGTCCACGCCGTTCGTGCGCCGGTGA 60
6 GTTCCAGGAGGTCGTGGAACTGGCCGTGAACGTCACCGCAAACGGTCACCGGCGCACGAA 60
7 TCCACGACCTCCTGGAACTCTTCAAAATCGGTGGCCTCCCACCGGACACCAACTATCTGT 60
8 TCAACAGAGTAGTAACCACGGTCAACGTAGTCACCCATGAACAGATAGTTGGTGTCCGGT 60
9 CCGTGGTTACTACTCTGTTGAAACCGTTACTCTCCTGCTCCTGTACAAACTGCGCTACCC 60
10 GACGAGACTCATGGTTACCACGCAGCAGATGCAGACGTTGCGGGTAGCGCAGTTTGTACA 60
11 GTGGTAACCATGAGTCTCGTCAGATCACCCAGGTTTACGGCTTCTACGACGAATGTATTC 60
12 GGTGAAGATGGTCCAAACGTTCGCAGAGCCGTATTTACGAATACATTCGTCGTAGAAGCC 60
13 ACGTTTGGACCATCTTCACCGACCTGTTCGACTACCTGCCGCTGACCGCGCTGGTTGAAA 60
14 TCAACGGTCGGAGAGAGACCACCGTGCAGGCAGAAAATGTCGTTTTCAACCAGCGCGGTC 60
15 GTCTCTCTCCGACCGTTGACACCTTCTCTCACATCCGTAATCTGGACCGTGTTCAGGAAG 60
16 GTCAGACCAGAGCAGGTCACACATCGGACCCTCGTGCGGAACTTCCTGAACACGGTCCAG 60
17 TGACCTGCTCTGGTCTGACCCGGACGACCGTGACGGTTGGGGTATCTCCCCGCGTGGTGC 60
18 TTGTGACAGAAACCCTCGGTAACACCCTGGCCGAAGGTAAAGCCCGCACCACGCGGGGAG 60
19 CCGAGGGTTTCTGTCACAACAACAAAATCAAAACCATCGCGCGTGCTCATCAGCTCGTTA 60
20 CGTAACCAGCTGATCCTGGTGGGTCCAAGAATAACCGTCCATAACGAGCTGATGAGCACG 60
21 CCAGGATCAGCTGGTTACGATCTTCTCTGCGCCTAACTACTGCTACCGTTGTGGCAATCT 60
22 AGCACTTATTCATGTGTTCGTCCAGCTCCAGCAGACCCGCGAGATTGCCACAACGGTAGC 60
23 GACGAACACATGAATAAGTGCTTCTTTCAGTTCGACCCTGCACCTCGTCGTGGCGAGGCA 60
24 TTACAGAAAGTAGTCCGGAGTCTTTTTAGAAACTTGTGCCTCGCCACGACG 51
Nanodrop:
The 260/280nm ratio is near 1.9, which suggests almost no protein contamination. The 260/230nm ratio is close to the desired 2.1, suggesting that there is little other contaminants at the 230nm absorbance.
The 260/280nm ratio is near 1.9, which suggests almost no protein contamination. The 2.09 value for the260/230nm ratio is close to the desired 2.1, suggesting that there is little other contaminants at the 230nm absorbance.
Daniel - results? - Dr. B 090913